作者：刘福强作者单位：辽宁医学院附属第一医院内分泌科 - h0 M: ?3 i/ M5 x4 _
) f' `# o! }7 ` u' S( F4 I2 b
J1 k( X6 o( {7 b. F8 h9 V * a# q6 }/ h1 z) _! b- f% n: R! }) d
, J- t% H" x7 J; m& y 7 w6 K9 d$ t; I) L# a
; h/ s6 [+ c; ]8 h
# ^& X+ X) y f8 {/ ? g6 ? + `) u6 b$ `0 }, o 0 J6 G3 f4 s3 l: j2 A & ~( [7 p9 G& T
2 f2 P* b0 j0 b' g$ ]( g 【摘要】 目的 探讨骨骼肌源性干细胞体外培养、鉴定的方法。方法 采用I型胶原酶和胰蛋白酶两步消化法分离大鼠骨骼肌源性干细胞，经差速贴壁法纯化后，培养液中培养。倒置显微镜观察细胞形态，生长曲线分析肌源性干细胞的增殖能力，结蛋白细胞免疫荧光方法鉴定肌源性干细胞。结果 经过两步消化和差速贴壁后， 成功培养出高纯度的肌源性干细胞，细胞免疫荧光结果为desmin( )。结论 通过体外原代培养获得高纯度肌源性干细胞，结蛋白细胞免疫荧光可以对其早期鉴定，为组织工程的临床应用提供种子细胞。 . w+ }0 N" f; Q' m7 ]0 }4 c9 @" }2 ]
【关键词】大鼠 肌源性干细胞 体外培养 6 {5 c* W" j3 g1 i/ A- A Culture and Identification of Rat Muscle Derived Stem CellsLIU fuqiang1, LIU Chang1, YANG Biao2, MEI Xifan23 ^: v4 g1 i) x
( H' F5 X$ e7 N- ~' o& } y
(1.Department of Endocrine, the First Affiliated Hospital of Liaoning Medical University;( G: r1 L& {( ]; p! N5 Q% G2 y
9 h. a; U8 f- t1 h* T% Q9 S
2.Department of Orthopaedics, the First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 China)Abstract：ObjectiveTo establish the methods for culture and identification of rat muscle-derived stem cells in vitro.MethodsBy two-step method, the rat muscle was digested with type I collagen and trypsin, and the isolated cell suspension was purified by different adhesion time . The proliferation ability of muscle-derived stem cells was analysed through studying growth curve, and the obtained cells were identified with cell immunofluroescence technique．ResultsHighly purified MDSCs were harvested and they showed desmin( ) by cell immunofluroescence technique．ConclusionsMDSCs can be cultured in vitro and can be identified by desmin stain and used to tissue engineering as “seed”cells.7 ]) n* Q. F; n% T' ?
; C' G8 Q6 Q2 ~' [# v2 L8 d ［2］ Torrente Y，Camirand G，Pisati F，et a1.Identification of a putative pathway for the muscle homing of stem ceils in a muscular dystrophy model［J］.J Cell Biol，2003，162(3)：511-520. # ]6 T/ J& b1 D3 J) e 9 i% ^1 V) `8 _3 y6 \& M! l ! r8 Z8 k+ }* L# g. i5 T : l- {: I o) V7 q% k ［3］ Cao B，Bruder J，Kovesdi I，et a1.Muscle stem cells can act as antigen-presentingcells.implication for gene therapy［J］.Gene Ther，2004，11：1321－1330. $ t' m/ M# C5 L& o, L3 ~7 Z5 E / g/ |0 Y; `$ a1 W( O; [' V u9 ^( h: v, M/ V# X* w' k% g% Z7 S2 V
+ L5 }9 x) r& V$ L- h a! @ ［4］ Thomas A ， R ， Helen M ， B. Primary mouse myoblast purification ， characterization ， and transplantation for cell mediated gene therapy［J］. J Cell Bio ，1994 ，125 :12758 l9 d" U8 _1 F7 f, y$ p4 X$ t
4 B& K x8 }. N! {& T- c# l 5 n2 v. p. \0 Z; n9 Y: x8 w ( ~/ D: p8 w" m) F# o ［5］ Zhuqing Qu，Deaaasy B，Jankowski R，et al.Identification of anovel population of muscle stem cells in mice:potential for Muscle regeneration ［J］.J Cell Biol，2002，(157):851-864." D. k3 U2 I9 f j- t* i" ^3 G" l4 f
, S# l9 H9 o- W, ~7 |2 u" d: q! v y/ C: }, V @9 W, r3 z9 E
& g _, W2 V1 ^! G# A9 j ［6］ Lee JY，Qu-Petersen Z，Cao B.et a1.Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing［J］.J cell Biol，2000，150，1085-l100. , O* e2 [) o' |5 H( i8 n! {% b4 Y3 t+ W4 N. U
& p8 Z2 M3 |6 n% `, E! g* f' R" f; O( Q' K8 ?
［7］ Lee JY，Cannon TW，Pruehnie R，et a1.The effects of periurethral muscle-derived stem cell injeetion on leak point pressure in a rat model of Stress urinary Inoonth lce［J］.Int urogynecol J，2003，l4：3l-37. 7 @% z: h2 ?# ?% {8 K& t# c( L# R/ M( G
% ^/ n! A2 K& [. K( y+ Z, x r! o0 z1 }/ C% H
［8］ Deasy BM，Jankow ski RJ，Huard J.M usele-derived stem cells：characterization and potential for cell-mediated therapy［J］.Blood Cells Molecules Diseases，2001，27：924-933. - e4 _4 Y- I1 B5 N1 n' [" u' g4 D* A, H+ H
; a% t8 e/ N1 V3 b7 S. @( \- \ 1 b" Z# q- S4 v; j/ j& \& o# Y0 F ［9］ Baroffio A ，Piallat MLB ，Gabbiani G ，et al. Heterogeneity in the progeny of single human muscle satellite cells［J］ . Differentiation ，1995，59(4) :259! n" i) I5 O* A! Y
& z/ B1 k, y# ^% i+ C N7 Z; _/ d+ _( b; P- C
% _" x) \% a1 C0 u9 D ［10］ Qu Z，Balkir L.Van Deutekom jc，et a1.Deve1o-pment of approaches toimprove cell survival in myoblast transfer therapy［J］.J Cell Biol，1998，142：1257-1267.作者: dypnr 时间: 2015-5-25 07:43